WO2006016693A1

WO2006016693A1 - Hemanalysis apparatus and method of hemanalysis

Info

Publication number: WO2006016693A1
Application number: PCT/JP2005/014882
Authority: WO
Inventors: Yasuhiro Horiike; Akio Oki
Original assignee: National Institute For Materials Science
Priority date: 2004-08-09
Filing date: 2005-08-09
Publication date: 2006-02-16
Also published as: US20080028821A1; JP4478776B2; JP2006052949A; US7678577B2; CN101006340A; DE112005001929T5

Abstract

A hemanalysis apparatus comprising a base plate; a hemocyte plasma separation part disposed on a lower end side of the base plate; a sensor part disposed on an upper end side of the base plate in relationship communicating with the hemocyte plasma separation part; a calibration solution reservoir disposed below the sensor part; and a waste calibration solution reservoir disposed above the sensor part. A first centrifugal axis is positioned above a hemocyte fraction accommodation portion of the hemocyte plasma separation part but below an upper end of a plasma fraction accommodation portion thereof. A second centrifugal axis is positioned inside the sensor part or at a locality proximal thereto. In the transfer/discharge of the calibration solution, the centrifugal force applied to the sensor can be reduced by carrying out low-revolution centrifugation round the first centrifugal axis remote from the sensor part. In the high-revolution centrifugal operation for hemocyte separation, the centrifugal force applied to the sensor part can be reduced by carrying out centrifugation round the second centrifugal axis. By such centrifugal operations, there can be accomplished hemocyte plasma separation and transfers of plasma and calibration solution as well as secure discharge of the calibration solution from the sensor to thereby realize high-precision analysis. Any sensor damaging attributed to strong centrifugal force at hemocyte plasma separation is avoided.

Description

Technical field of blood analyzer and blood analysis method

The present invention relates to a blood analyzer constituted by an ultra-small groove channel manufactured on an insulating material substrate such as a quartz plate or a polymer resin plate. In particular, when a small amount (less than a few liters) of blood is introduced into the groove channel on the substrate and centrifuged to separate blood cell components and plasma components, the concentration of various chemical substances in the plasma components is measured. In addition, the present invention relates to a flow path and a substrate structure for carrying a liquid such as an analytical sensor calibration liquid or blood by centrifugal force. Background art

Conventional health examinations and diagnosis of disease states have been carried out by collecting a large amount of blood of several C C from patients and analyzing them using measurements obtained with a large-scale automated blood analyzer. Normally, such automatic analyzers are installed in medical institutions such as hospitals, are large in scale, and their operations are limited to those with specialized qualifications.

However, in recent years, by applying the microfabrication technology used for the production of semiconductor devices that have advanced extremely recently, analyzers such as various sensors are placed on a substrate that is at most several cm to several cm square, and the blood of the subject, etc. There is a growing interest in the development and practical application of new devices that can guide body fluids and instantly understand the health status of subjects. With the emergence of such low-cost devices, health insurance benefits, which are steadily increasing, can be reduced by enabling daily health management of elderly people at home in the coming aging society. Also, in the field of emergency medicine, various social effects such as the appropriate response can be made if the device can be used to quickly determine the presence or absence of infectious diseases (such as hepatitis, acquired immune deficiency). Expected to be non It is a technical field that is always drawing attention. Thus, instead of the conventional automatic analyzer fc, a small and simple blood analysis method and a blood analyzer aiming to carry out blood analysis by one's own hands in each home have been developed (for example, Patent Document 1). reference).

Patent Document 1: Japanese Patent Application Laid-Open No. 2 00 8-2 8 8 8 6

FIG. 1 shows an example of a micro-module blood analysis device described in Patent Document 1. Reference numeral 101 denotes a lower substrate of the blood analyzer, and a fine groove channel (micro-cavity) 10 2 formed by etching on the lower substrate is provided. On this lower substrate 1001, an upper substrate (not shown) of substantially the same size is bonded, and the groove flow channel 102 is sealed from the outside.

In the flow path 102, blood sampling means 103, plasma separation means 104, analysis means 105, and moving means 106 are sequentially provided from the most upstream part to the most downstream part. A hollow blood collection needle 10 3 a is attached to the blood collection means 10 3 at the forefront of the flow path, and this needle 10 3 a is inserted into the body to serve as an inlet for blood into the substrate. Separating means 10 4 is a curved portion of the flow path 10 2, and is made of, for example, a U-shaped micro-cabinet. After the collected blood is guided to the U-shaped microphone mouth cavity, the substrate is subjected to acceleration in a certain direction by a centrifuge, thereby precipitating blood cell components at the bottom of the U-shaped part and plasma as the supernatant. Isolate. The analysis means 10 5 is a sensor for measuring each concentration of blood such as pH value, oxygen, carbon dioxide, sodium, potassium, calcium, dulcose, and lactic acid.

The moving means 10 6 located at the most downstream part of the flow path is for moving blood by electroosmotic flow in the micro-chamber, and the flow path part 1 connecting between the electrodes 1 0 7 and 1 0 8. It consists of 0-9. The buffer solution filled in the flow path is moved to the downstream side of the flow path by the electroosmotic flow generated by applying a voltage between the electrodes. Blood is taken into the substrate from 0-3. In addition, plasma obtained by centrifugation To the analysis means 1 0 5.

1 1 0 is an output means for extracting information from the analysis means, and is composed of electrodes and the like. 1 1 1 is a control means for controlling the above collection means, plasma separation means, analysis means, moving means, and output means as required. The blood collected from the sampling means 10 3 is separated into plasma and blood cell components by the separation means 10 04, and this plasma is led to the analysis means 10 5, where the pH value, oxygen, Measure concentrations of carbon, sodium, potassium, calcium, dulcose, urea nitrogen, creatinine, and lactic acid. Movement of blood between each means is performed by moving means 106 having a pumping capacity such as those using phenomena such as electrophoresis and electroosmosis. In FIG. 1, the downstream area of the flow path 102 is divided into five parts, each of which is provided with analysis means 10 5 and moving means 10 6.

A glass material such as quartz is often used for the substrate of such a blood analyzer, but it is more suitable for manufacturing a large amount of the device at a low cost, and in consideration of disposal when disposable. In recent years, resin materials have come to be used.

In the conventional blood analyzer shown in FIG. 1, a moving means such as an electroosmotic pump 10 6 is required when a blood sample is introduced into the apparatus. After the introduced blood is centrifuged together with the substrate to obtain plasma, it is necessary to operate the electroosmotic pump 10 6 again to move the plasma to the analysis means 10. In addition, when the analysis means is a sensor configured based on an electrochemical principle, it is necessary to calibrate the sensor using a calibration solution in advance. That is, before introducing plasma to the sensor, the sensor must be immersed in the calibration solution to calibrate the sensor, and the calibration solution after calibration must be discharged from the analysis means. A moving means such as a pump is also required for transferring the calibration liquid.

As the moving means, it is conceivable to use an electroosmotic pump provided in the same substrate as shown in Fig. 1 or a negative pressure pump installed outside the substrate. These moving means move blood, plasma, and calibration liquid by pumping or sucking them. At this time, in order to move a desired liquid to a desired position in the blood analyzer, it is necessary to accurately control the suction force of the moving means. For this purpose, a liquid position sensor must be newly installed in or outside the blood analyzer, and there is a problem that the apparatus becomes expensive because of the addition of these control mechanisms and position sensors. .

If the analysis means is a sensor constructed based on electrochemical principles, the sensor is calibrated with a calibration solution (standard solution) containing a known concentration of the test component, and then the calibration solution is discharged from the analysis means. Must. However, even if the calibration liquid is discharged, some calibration liquid remains on the surface of the analysis means and the flow path means according to the wettability of the surface. As described above, the size of the means constituting the apparatus such as the flow path means is the size of the blood analysis apparatus that is currently targeted in order to analyze the concentration of various chemical substances present in a minute amount of blood of several microliters. It is getting smaller. In general, as the size of an object decreases, its surface area (S) to volume (V) ratio S / V increases, which means that the surface effect becomes prominent. Therefore, even if the amount of calibration liquid remaining on the flow path means or the analysis means surface is small, an analyzer that introduces a small amount of plasma has a problem that the concentration of the measured chemical substance varies. For this purpose, it is necessary to ensure that the calibrated calibration solution is discharged from the analysis means before introducing the plasma into the analysis means.

In view of such circumstances, the present inventors are a blood analyzer that separates plasma in a flow path by a centrifugal operation, and transports blood, plasma, and calibration fluid within the apparatus without using a pump or the like. In addition, a blood analysis device has been proposed that enables high-accuracy analysis by reliably discharging the calibration liquid from the sensor portion (see, for example, Patent Document 2).

Patent Document 2: Japanese Patent Application 2 0 0 3— 0 4 0 4 8 1

FIG. 2 shows an example of a blood analyzer described in Patent Document 2 (unpublished). Reference numeral 2 0 1 is an upper substrate on which a flow path is formed, and 2 0 2 is a sensor This is the lower substrate on which the electrodes 20 3 and electrode terminals 2 0 4 for taking out sensor signals to the outside are formed. A blood collection needle 2 0 5 is attached to the upper substrate 2 0 1, and the collected blood is sucked into the blood reservoir 2 0 7 through the guide flow path 2 0 6 and external pump (not shown) from the pressure port 2 0 8 Move by. The flow path 2 0 9 and the flow path 2 1 0 are connected to the opening holes 2 1 1 and 2 1 2 provided on the side walls of the upper substrate 2 0 1, respectively. The opening holes 2 1 1 and 2 1 2 are closed by a holder (not shown) for attaching the. Similarly, the calibration liquid reservoir 2 1 3 contains the calibration liquid injected from the suction / pressure feeding port 2 08.

An example of the operation of this proposed blood analyzer substrate will be described next. First, when the blood analyzer substrate is centrifuged around the first centrifugal force central axis 2 1 4, the calibration liquid in the calibration liquid reservoir 2 1 3 passes through the guide channels 2 1 5 and 2 1 6, It is carried into a plurality of sensor grooves 2 1 7 containing a plurality of sensors 2 0 3. After calibrating sensor 203, turn the blood analyzer substrate 90 degrees clockwise and place it on the centrifuge. That is, when the substrate is centrifuged around the second centrifugal force central axis 2 1 8 located on the left side in FIG. 2, the calibration groove fills the sensor groove 2 1 7 and the calibration liquid 2 1 6 and 2 1 9 Is stored in the calibration liquid waste reservoir 2 20.

Thereafter, the blood analyzer substrate is rotated 90 degrees counterclockwise and placed on the centrifuge. That is, when the substrate is moved away from the center of the first centrifugal force central axis 2 14, blood is transported from the blood reservoir 2 07 to the sensor groove 2 17 via the guide channel 2 2 1. If centrifugal force is continuously applied in this state, the blood cell component in the blood is fractionated in the direction in which gravity is applied, that is, below the sensor groove 2 17, and the plasma component is the supernatant above the sensor groove 2 17. As isolated. Since the sensor group 20 3 is arranged in that area, a plurality of electrode terminals in which pH values in blood, oxygen, carbon dioxide, sodium, potassium, calcium, glucose, lactic acid, etc. are connected to each sensor. Measured by an external measuring instrument via 2 04. This proposed blood analyzer is capable of centrifugal operation in two different directions, and the calibration liquid in the calibration liquid reservoir is conveyed to the sensor unit by the centrifugal operation in the first centrifugal direction, and after the sensor calibration Is designed to ensure that the calibration solution can be discharged from the sensor section by centrifuging in the second centrifugal direction. After the calibration solution is discharged, the blood in the blood reservoir can be transported to the sensor unit and separated into blood cells and plasma by centrifuging in the first centrifugal direction.

However, while having these advantages, a problem that cannot be ignored to perform blood analysis in a short time due to the use of centrifugal force.

Needless to say, it is important that the measurement time with the blood analyzer chip is as short as possible. In this hematology analyzer, the distance from the center axis of the centrifuge to the center of the chip is 5 cm, and the time required for injecting and discharging the calibration liquid is usually low centrifugal force of 300 rpm or less. But it is about 1 second. However, in order to separate blood cells and plasma in blood in a few seconds to several minutes, a centrifugal force of at least 400 rpm is required in the blood cell separation region. Fig. 12 shows the relationship between the rotation speed (rpm) and acceleration (G) at this time. Gravitational acceleration of 5 0 0 G at 3 00 rpm and 1 0 0 0 at 4 0 0 0 rpm Equivalent to applying G gravitational acceleration.

It was found that the output of the sensor decreased due to the centrifugal operation during blood cell / plasma separation. For example, looking at the output voltage when a calibration solution (containing 137 mM sodium ions) was measured with a sodium ion sensor, it was affected by the rotational speed (r p m) as shown in Fig. 3. Up to about 300 rpm, the sensor output shows a stable value of about 20 O mV, but at higher speeds, the sensor output shows a decreasing trend and the variance of the value increased. . In this measurement, a sensor showing a stable value of about 20 O mV up to 10 00 r pm was prepared and used for each rotation experiment. Although not specifically shown, the same tendency was also found in the measurement of force rion ions.

The sodium ion concentration sensor captures sodium ions. An ion sensitive membrane Bis (12-crown-4) and an anion exclusion agent that serves to prevent the penetration of plasma anions (anions) into the sensitive membrane are mixed with PVC (polyvinyl chloride). This is fixed on the carbon electrode to form a sensor. At that time, a large amount of plasticizer is mixed with PVC to make it easier to incorporate sodium ions into the sensitive membrane. If the centrifugal force at 7000 rpm is estimated from the weight per sensor, the force applied to the sensor is about pico-two tons. However, the cause of the decrease in sensor output at this high rotational speed is that the PVC film containing plasticizer containing this ion-sensitive film is deformed on the carbon electrode by a strong centrifugal force, and a part of the PVC film is carbon. It is assumed that it was peeled off from the electrode and water was infiltrated. Although it may be possible to change the composition of the film to harden the film and to fix the film to the carbon electrode, curing the film loses the characteristics of the original electrochemical sensor. Disclosure of the invention

The present invention has been made in view of such circumstances, and is a blood analyzer that separates plasma by a centrifugal operation, and transports plasma and calibration fluid within the apparatus without using a pump or the like. The first is to provide a blood analyzer that can discharge the calibration liquid from the sensor part reliably, and can perform more accurate analysis without damaging the sensor due to centrifugal operation during plasma separation. The purpose.

Further, according to the present invention, when a blood analyzer that separates plasma by centrifugation is used, plasma and calibration fluid can be transported in the device only by centrifugation, and the calibration fluid is reliably discharged from the sensor portion. In addition, a second object is to provide a blood analysis method that enables highly accurate analysis without damaging the sensor due to centrifugation during plasma separation.

According to the present invention, a first object is to provide a blood analyzer for performing plasma separation of a whole blood sample by centrifugation and analyzing a test component in a blood liquid component: (a) A blood cell disposed on the lower end side of the substrate. A plasma separation unit, a blood cell fraction storage unit that deposits and stores a blood cell fraction when centrifugal force is applied, and a plasma fraction that is located above the blood cell storage unit and stores plasma A blood cell / plasma separation unit equipped with a storage unit;

(b) a sensor unit disposed on the upper end side of the substrate, the sensor unit having a sensor groove containing a sensor for analyzing a test component;

(c) a blood cell / plasma guide channel communicating the plasma separation part and the sensor part;

(d) a blood inlet for introducing a whole blood sample into the blood cell and plasma separator;

(e) a calibration fluid reservoir containing calibration fluid for calibrating the sensor;

(f) a calibration liquid waste reservoir containing calibration liquid after sensor calibration;

(g) a calibration fluid introduction flow path communicating with the calibration fluid reservoir and the sensor groove;

(h) a calibration fluid discharge channel that communicates the sensor groove with the calibration fluid waste reservoir;

While it is possible to centrifuge around the first centrifugal axis located above the blood cell fraction storage unit and below the upper end of the plasma fraction storage unit; inside or close to the sensor unit than the blood cell / plasma separation unit The calibration liquid reservoir is located below the sensor unit and above the first centrifugal axis, and the calibration liquid waste liquid reservoir is the sensor. This is achieved by a blood analyzer characterized by being located above the section.

In other words, the blood analyzer of the present invention is capable of being centered around two different centrifugal shafts, and transporting calibration fluid and waste fluid is centrifuged around the first centrifugal shaft away from the sensor unit. Although the radius increases, the gravity acceleration applied to the sensor can be reduced by centrifuging at a low speed. On the other hand, in centrifugal operation that gives a large gravitational acceleration for blood cell separation, the centrifugal axis (second centrifugal axis) at this time is placed inside or close to the sensor unit so that the gravitational acceleration applied to the sensor unit is small. By setting the position, even if centrifugal force is applied to the blood cell / plasma separation part, large gravitational acceleration is not applied to the sensor part. This It is possible to prevent the sensor from being damaged due to excessive centrifugal force.

Blood cell here. In a preferred embodiment, the plasma separation part is formed by a U-shaped flow path, the blood cell fraction storage part is provided at the lowermost curved part, and the upper part is used as the plasma fraction storage part. The blood cell fraction storage part may be provided so as to protrude downward from the lowermost end of the u-shaped flow path, and in that case, the volume of the blood cell fraction storage portion in the whole blood sample introduced into the U-shaped flow path is provided. It is preferable to make it larger than the stroke amount. The blood inlet can be provided on the side wall of the U-shaped channel above the plasma fraction container.

The U-shaped channel is preferably provided with an air vent channel so that the whole blood sample can be smoothly introduced to the lowermost end of the U-shaped channel. The most preferable mode is to communicate with the lowermost end of the channel.

A plurality of sensor grooves may be provided in the sensor unit, and a plurality of sensors for analyzing different test components may be accommodated in each sensor groove. In this case, if the sensor grooves are arranged on the circumference and the center is the second centrifugal axis, that is, if the sensor grooves are arranged radially around the second centrifugal axis, the second centrifugal axis is the center. When blood cells and plasma are separated by centrifugal operation, the distance between the sensor and the centrifugal center is the shortest, and the gravitational acceleration applied to the sensor can be minimized. The blood introduction port may be provided with a blood collection mechanism that stores already collected blood in the blood introduction port. By hydrophilizing the plasma inlet and blood cell / plasma separator, blood samples can be introduced and plasma can be transported smoothly. Similarly, if the plasma guide flow path, sensor groove, calibration liquid reservoir, calibration liquid waste liquid reservoir, calibration liquid introduction flow path, and calibration liquid discharge flow path are each hydrophilized, the calibration liquid transport and the plasma transport can be performed. Become smoother.

The second object of the present invention is a blood analysis method comprising the following steps: (1) A blood cell / plasma separation unit disposed on the lower end side of the substrate, and the blood cell fraction when centrifugal force is applied A blood cell fraction containing part for precipitating and containing the blood cell, and a plasma fraction containing part for storing plasma located above the blood cell fraction containing part. A blood cell> a plasma separation unit; a sensor unit having a sensor groove which is disposed on the upper end side of the substrate and analyzes a test component; and a blood plasma guide channel communicating the blood cell plasma separation unit and the sensor unit A blood introduction port for introducing a whole blood sample into the blood cell / plasma separator; a calibration liquid reservoir for storing a calibration liquid for calibrating the sensor; a calibration liquid waste reservoir for storing a calibration liquid after sensor calibration; A blood analyzer having a calibration liquid introduction flow path that communicates between the liquid reservoir and the sensor groove; and a calibration liquid discharge flow path that communicates between the sensor groove and the calibration liquid waste liquid reservoir;

(2) Centrifuge the blood analyzer around the first centrifugal central axis located above the blood cell fraction container and below the upper end of the plasma fraction container, and remove the calibration solution in the calibration reservoir. Introduced into the sensor groove;

(3) calibrate the sensor;

(4) The blood analyzer is centrifuged around the first centrifugal axis, and the calibration solution in the sensor groove is discharged into the calibration solution reservoir;

(5) By introducing a whole blood sample into the blood cell / plasma separation unit and centrifuging the blood analyzer around the second centrifugal axis located closer to the sensor unit than the blood cell / plasma separation unit, Blood cell / plasma separation is performed in the blood cell / plasma separator to precipitate the blood cell fraction in the blood cell compartment;

(6) centrifuge the blood analyzer around the first centrifuge central axis to convey the plasma fractionated in the plasma fraction storage unit to the sensor groove;

(7) The analysis of the liquid component in the plasma in the sensor groove is performed by a sensor.

Gravity acceleration applied to the sensor in the calibration liquid introduction process in step (2), the calibration liquid discharge process in step (4), and the plasma transport process in step (6) in which centrifugation is performed around the first centrifugal axis. Is preferably 500 G or less. In addition, the step of centrifuging around the first centrifugal axis (5)

In the blood cell / plasma separation step, it is preferable that the gravitational acceleration applied to the plasma separation unit is 1000 G or more, while the gravitational acceleration applied to the sensor is 500 G or less. If an air vent channel is provided in the plasma fraction storage part of the blood analyzer, outside air is pressurized from the air vent channel without performing a centrifugal operation in the plasma transfer step of step (6). It may be introduced to transport plasma to the sensor unit. Brief Description of Drawings

FIG. 1 is a conceptual diagram showing an example of a conventional micro-module blood analyzer.

FIG. 2 is an overall perspective view of a blood analyzer (unknown) proposed by the inventors.

FIG. 3 is a graph showing that the output voltage when the calibration solution (containing 137 mM sodium ion) is measured by a sodium ion sensor varies with the number of revolutions (r p m).

FIG. 4 is a schematic plan view of a blood analyzer according to an embodiment of the present invention. FIG. 5 is a schematic diagram showing another example of arrangement of sensor grooves.

FIG. 6 is a schematic diagram showing still another example of arrangement of sensor grooves.

FIG. 7 is a diagram showing the state of the blood analyzer on the rotator in the calibration liquid introduction process and the calibration liquid discharge process in one embodiment of the blood analysis method of the present invention.

FIG. 8 is also a diagram showing the state of the blood analysis device on the rotator in the blood cell / plasma separation process.

FIG. 9 is also a diagram showing the state of the blood analyzer on the rotator in the plasma transfer process.

FIG. 10 is a diagram showing parameters for obtaining the number of rotations when the solution is discharged from the thin tube by centrifugal force.

Fig. 11 shows various parameters for calculating the rotational speed for introducing the calibration liquid by centrifugal force and the rotational speed for discharging the calibration liquid through the mechanical valve in the blood analyzer of the present invention. It is a figure which shows evening. Figure 12 shows the relationship between the gravitational acceleration (G) and the number of rotations (rum) that occur when a rotating body with a radius of 50 nm is rotated.

In addition, the code | symbol in a figure shows the following.

1 0 Blood analyzer (substrate)

1 2 U-shaped channel (blood cell / plasma separator)

1 Blood cell reservoir (Blood cell compartment)

1 6 Plasma fraction storage

1 8 Blood inlet

2 0 Blood collection mechanism

2 8 Air vent channel

2 9 Air vent hole

3 0, 3 0 A, 3 0 B Sensor section

3 2, 3 2 A, 3 2 B Sensor groove

3 4 Sensor

3 8 External electrode terminal

4 0 Plasma guide channel

4 2 Calibration solution reservoir

4 4 Calibration solution introduction flow path

4 6 Calibration solution waste reservoir

4 8 Capillary valve (Calibration solution discharge flow path)

5 0, 5 2 Air escape passage

6 0 Rotator

6 2 Board guide groove

C 0 Rotator center axis

C 1 First centrifugal axis (centrifugal force center)

C 2 Second centrifugal axis (centrifugal center) BEST MODE FOR CARRYING OUT THE INVENTION FIG. 4 is a schematic plan view of a blood analyzer according to an embodiment of the present invention. Reference numeral 10 denotes a substrate of the blood analyzer formed vertically in the figure, which is a superposition of the substrate on which the flow path is formed and the substrate on which the sensor electrode and wiring are formed. Indicates. The upper and lower substrates are made of, for example, a resin such as polyethylene television (PET) or polycarbonate (PC). Inside the substrate 10, a blood cell / plasma separation unit 12 having a U-shaped flow path is disposed on the lower end side thereof, and a blood cell reservoir 1 serving as a blood cell fraction storage unit 1 is disposed at the lowermost curved portion thereof. 4 is provided. The upper part of the blood cell reservoir 14 is a plasma fraction container 16 where plasma is fractionated as a supernatant during centrifugation. 1 5 is a blood cell reservoir (blood cell compartment) 1 4 is a backflow stopper to prevent blood cells that have settled in 4 from flowing back when handling the substrate. Fig. 4 shows the state after the blood cell / plasma separation operation, and the blackened portion in the blood cell reservoir 14 shows the fractionated blood cells. The hatched portion of the plasma fraction storage part indicates the fractionated plasma.

U-shaped channel 1 2 Side wall is provided with a blood inlet 18 for introducing a whole blood sample above the plasma fraction container 16 and a blood collection mechanism 20 storing the collected blood can be installed here It has become. The blood collection mechanism 20 is composed of a stainless painless needle 2 2, a reinforcing stainless steel tube 24, and a primary blood reservoir 26 for storing blood after blood collection. Inserted into blood inlet 1 8. 28 is an air vent channel communicating with the lowermost end of the U-shaped channel, facilitating the introduction of a whole blood sample from the blood inlet 18. The first centrifugal axis C 1 is located above the blood cell fraction storage unit 14 and below the upper end of the plasma fraction storage unit 16. The plasma fraction above the centrifugal axis C 1 is conveyed to the sensor unit 30 described later by centrifugal operation. Therefore, the position of the first centrifugal axis C 1 is determined according to the amount of plasma fraction to be conveyed.

The sensor unit 30 is arranged on the upper end side of the substrate 10 and has a plurality of sensor grooves 32 arranged radially around the second centrifugal axis C2. Each sensor groove 3 2 contains a sensor 3 4, and the output of each sensor is It is led to each electrode terminal 38 exposed to the outside of the substrate through the wiring. The sensor 34 includes, for example, an electrode such as silver / silver chloride or carbon, and a silver / silver chloride reference electrode. The wiring is made of, for example, silver-containing carbon, and the outer electrode 38 is made of, for example, silver. These silver / silver chloride, carbon electrode, silver / silver chloride reference electrode, silver-containing carbon wiring, silver electrode, etc. are formed by screen printing, for example.

40 is a plasma guide channel that collects the upper part of the U-shaped channel 12 and communicates with the sensor unit 30, and the plasma fractionated in the plasma fraction storage unit 16 after the blood cell / plasma separation operation To the sensor unit 30. Reference numeral 42 denotes a calibration liquid reservoir that contains a calibration liquid for calibrating the sensor, and is connected to the sensor unit 30 by a calibration liquid introduction channel 44. The calibration liquid reservoir 42 is located below the sensor unit 30 and above the first centrifugal axis C1. Therefore, when the substrate 10 is centrifuged about the first distal axis C 1, the calibration liquid in the calibration liquid reservoir 42 is conveyed to the sensor unit 30.

A calibration solution waste liquid reservoir 46 is provided above the sensor unit 30 and communicates with the sensor unit 30 via a calibration solution discharge channel 48 (an exhaust valve described later). In FIG. 4, the calibration liquid waste reservoir 46 is disposed so as to surround the sensor unit 30, but at least the volume above the sensor unit 30 is larger than the amount of calibration liquid discharged here. A large volume is sufficient. Reference numerals 50 and 52 are air vent channels.

In the present embodiment, the sensor grooves 3 2 in the sensor section 30 are arranged radially, but as shown in FIG. 5, the sensor grooves 3 2 A are arranged side by side around the second centrifugal axis C 2. 3 OA may be formed. Further, as shown in FIG. 6, the outer wall of the sensor part 30 B may be rectangular, and the sensor grooves 32 B may be arranged radially inside the sensor part 30 B. 4, 5, and 6, the second centrifugal shaft C 2 is arranged inside the sensor units 30, 3 OA, and 30 B. This is to make the heavy acceleration applied to the sensor as small as possible. This is because it is a convenient arrangement. The second centrifugal shaft C 2 is not necessarily arranged inside the sensor unit 30, The gravitational acceleration applied to the sensor can be reduced if the position is close to the support 30.

A method of using this blood analyzer will be described with reference to FIGS. First, the sensor is calibrated before blood analysis. As shown in FIG. 7, the substrate 10 of the blood analyzer is placed in a guide groove 62 provided in the diametrical direction on the rotator 60 and placed on the upper side. Fix so that the first center axis C 1 of the substrate 10 is aligned with the C 0 position. When the substrate 10 is centrifuged in this state, the calibration liquid in the calibration liquid reservoir 42 is conveyed to the sensor unit 30. At that time, the air in the sensor unit 30 is exhausted from the air vent channel 52. At this time, the number of rotations of the centrifuge is set so that the calibration solution does not pass through the chiral pulp 48. Stop centrifuge and calibrate each sensor on rotator 60.

After the sensor is calibrated, the calibration solution in the sensor groove 32 is discharged. After the sensor calibration, the position of the substrate 10 is left as it is, the rotator 60 is rotated again, the substrate 10 is centrifuged, and the calibration solution in the sensor unit 30 is discharged to the calibration solution waste reservoir 46. This centrifuge operation can remove the calibration solution covering the sensor, eliminating the possibility of errors in the analytical value due to the residual calibration solution. In this calibration liquid discharging process, the calibration liquid is passed at higher speed than the centrifuging in the calibration liquid transporting process so that the calibration liquid passes through the chiral pulp 48. However, even in such a case, it is preferable that the acceleration of gravity is such that the sensor is not damaged by the centrifugal force, and the centrifugal force applied to the sensor unit 30 is preferably 50 G or less.

Next, whole blood sample introduction and blood cell / plasma separation are performed by centrifugation. The blood collection mechanism 20 is inserted into the blood introduction port 18 of the substrate 10, the substrate 10 in this state is moved downward in the guide groove 62, and the second centrifugal axis C 2 is rotated by the rotator 60. Fix it so that it matches the position of axis C0 (Fig. 8). When the substrate 10 is centrifuged in this state, the whole blood sample is conveyed into the U-shaped channel 12 and further separated into plasma and blood cells. The blood cell fraction is stored in the blood cell reservoir 14 and the plasma fraction is in the U-shaped flow path. It is fractionated as a supernatant in the upper part of 1 2 (plasma fraction accommodating part 16). The centrifugal operation at this time is performed in order to completely separate blood cells, and it is preferable that a centrifugal force of 100 G or more is applied to the lowermost part of the U-shaped channel.

After blood cell separation, the substrate 10 is again moved upward in the guide groove 62 and fixed so that the first distal axis C 1 coincides with the rotational axis C 0 of the rotator 60 (FIG. 9). ). When the substrate 10 is centrifuged in this state, the plasma above the centrifugal axes C 0 and C 1 shown in FIG. 9 is conveyed to the sensor unit 30 by centrifugal force. The blood cell reservoir 14 is below the centrifugal axes C 0 and C 1, and the blood cells fractionated here do not move to the sensor unit 30. The centrifugal operation at this time is preferably a gravitational acceleration that does not damage the sensor, and the centrifugal force applied to the sensor unit 30 is preferably 500 G or less. Finally, each test component in plasma is measured by each sensor.

The important point of this embodiment is that the centrifugal axis used in the calibration liquid introduction process and the centrifugal axis used in the calibration liquid discharge process are both the first centrifugal axis C 1 (FIG. 7). . When the calibration liquid is introduced from the calibration liquid reservoir 42 to the sensor unit 30, the calibration liquid transferred to the sensor unit 30 must not be further transferred to the calibration liquid waste liquid reservoir. In other words, the relatively weak centrifugal force used in the calibration liquid introduction process, the calibration liquid introduction flow path 4 4 and the flow path diameter of the chiral pulp (calibration liquid discharge flow path) 4 8 are estimated, and the calibrated calibration liquid is further used as the calibration valve. It is necessary to estimate the relatively strong centrifugal force that is transferred to the calibration liquid waste reservoir 4 6 through 4 8.

As regards mildew pulp, it is described on page 315 of “Fundamental and Applicat ions of Microfludicsj (Publisher: Artech House (Bos ton-London) 2002)” by Nam- Trung Nguyen and Steven T. Wereley. are. as shown in FIG. 1 0, the solution to the capillary is present between the centrifugal center of radius and radius R _2, the contact angle to a solution tubules when the solution is discharged from the capillary 0, § surface tension, The radius of the capillary is R, the density of the solution When the degree is P, the following relationship exists between the minimum number of rotations fm at which the solution tries to protrude from the capillary tube due to centrifugal force.

fm ² ≥ rcos0 / R · p · Γ ² · (R ₂ -Rj) (R ₂ + R ₂ ) As shown in Fig. 11, the distance between the first centrifugal axis C 1 and the second centrifugal axis C 2 is 5 The length of the flow path (R ₂ — R!) from the calibration liquid reservoir 42 through the flow path to the groove that houses the sensor (ie, the calibration liquid introduction flow path 44) is 1 cm, and the first centrifuge The distance from the axis C 1 to the sensor part 30 side end of the calibration fluid reservoir 42 is 3.5 cm. The surface tension (a) of water at 25 is 72X10 " ³ [N / m], and when the polyethylene terephthalate resin is used as the substrate 10 material, the contact angle with water Θ is 80 degrees. The density (p) is 1 Xl0 ³ [kg / m ³ ] Using these values, the diameter of the calibration liquid discharge channel 44 (2) even if the minimum speed (fm) is l OO r pm. R) is approximately 3 m or more, that is, if the calibration introduction flow path is 1 cm long and the diameter is greater than 100 m, the first centrifugal axis C 1 is centered at 100 rpm. Centrifugal solution can be transferred from the calibration solution reservoir 42 to the sensor unit 30 by centrifugation.

On the other hand, if the flow path length of the valve 48 (R ₂ — is 0.5 cm and the diameter (2 R) is about 100 // m, when the fm is about 100 00 rpm or more, the sensor section (sensor Calibration fluid in the groove) flows into the waste reservoir 46. Here, the gravitational acceleration applied to the sensor is about 60 G from Fig. 12 because the centrifugal radius to the sensor is about 5.5 cm. In the blood analyzer substrate manufactured with such a flow path dimension, both the calibration liquid and the plasma flow operated normally.

The advantage of this blood analysis substrate is that the sensor is disposed at a position on the radiation about 5 mm away from the second centrifugal axis C2. The distance from the first centrifugal axis C 1 is about 1/10 compared to about 5 cm. Compared with the case where the centrifuge is performed with the first centrifuge shaft C 1 as the center, the centrifuge with respect to the second centrifuge shaft C 2 receives only about 1/10 of the centrifugal force. Therefore, the centrifugal operation in the blood cell separation process shown in Fig. 8 can be performed even if the second centrifugal axis is centrifuged at 7 000 rpm. The sensor did not show any damage.

In the plasma transfer step, as shown in FIG. 9, the blood cell reservoir 14 is located on the opposite side of the plasma guide channel 40 across the centrifugal axis C 0 (first centrifugal axis C 1), and the blood cells Only plasma could be transported to the sensor section without backflowing into the plasma guide channel 40. In this embodiment, the substrate 10 is moved from the position shown in FIG. 7 to the position shown in FIG. 9 for transferring the plasma after blood cell separation. However, after blood cell separation, air is removed from the air vent hole 29 in the air vent flow path 28. If it is put and pressurized, the plasma can be conveyed to the plasma guide channel 40 and the sensor unit 30 (see FIG. 4). Industrial applicability

As described above, the blood analyzer of the present invention is capable of being centered around two different centrifugal axes. The calibration solution is carried into the sensor groove and discharged, and the blood cell is separated into the plasma sensor unit. Can be introduced by centrifugal force without using any pump. There is no need to use a conventional negative pressure pump, and an inexpensive and simple blood analyzer can be realized. Conveying calibration solution · Waste liquid can be reduced in gravity acceleration applied to the sensor by centrifuging at a low speed around the first centrifugal axis away from the sensor unit. On the other hand, in centrifugal operation that gives a large gravitational acceleration for blood cell separation, the gravitational acceleration applied to the sensor can be reduced by centrifuging around the second centrifugal axis. Therefore, the multi-layered sensor composed of different components is not damaged by the strong centrifugal force during blood cell separation, and more accurate analysis is possible.

Claims

The scope of the claims

1. In a blood analyzer that separates the whole blood sample by centrifugation and analyzes the test components in the blood fluid component:

(a) A blood cell / plasma separation unit disposed on the lower end side of the substrate, the blood cell fraction containing unit for precipitating and storing a blood cell fraction when a centrifugal force is applied, and a blood cell containing fraction A blood cell / plasma separation unit provided with a plasma fraction storage unit for storing plasma located above the unit;

(b) a sensor unit disposed on the upper end side of the substrate, the sensor unit having a sensor groove that accommodates a sensor for analyzing a test component;

(d) a blood inlet for introducing a whole blood sample into the blood cell / plasma separator;

(h) a calibration liquid discharge flow path that communicates the sensor groove with the calibration liquid waste liquid reservoir;

While being capable of being centrifuge around a first centrifuge shaft located above the blood cell fraction container and below the top of the plasma fraction container;

It is possible to centrifuge around the second centrifugal axis located in the sensor part or closer to the blood cell / plasma separation part;

The blood analyzer characterized in that the calibration solution reservoir is located below the sensor unit and above the first centrifugal axis, and the calibration solution waste solution reservoir is located above the sensor unit.

2. The blood cell / plasma separation part is formed by a U-shaped flow path, the blood cell fraction storage part is provided at the curved part at the lower end of the U-shaped flow path, and the upper part thereof is a plasma fraction storage part. The blood analyzer according to claim 1 characterized by the above.

3. The blood cell fraction storage part protrudes downward from the bottom of the U-shaped channel. 3. The blood analyzer according to claim 2, wherein the blood analyzer has a volume larger than a blood cell fraction in the whole blood sample introduced into the u-shaped channel.

4. The blood analyzer according to claim 2, wherein the blood introduction port is provided on a side wall of the U-shaped flow path above the plasma fraction storage unit.

5. The blood analyzer according to claim 2, wherein the U-shaped channel is provided with an air vent channel communicating with the U-shaped channel.

6. The blood analyzer according to any one of claims 1 to 5, wherein the sensor section has a plurality of sensor grooves in which sensors for analyzing different test components are accommodated.

7. The blood analyzer according to claim 6, wherein the plurality of sensor grooves are arranged on a circumference around the second centrifugal axis.

8. The blood analyzer according to any one of claims 1 to 7, wherein the plasma inlet, the blood cell / plasma separator, the plasma guide channel, and the sensor groove are each subjected to a hydrophilic treatment.

9. The blood analyzer according to claim 8, wherein the calibration liquid reservoir, the calibration liquid waste liquid reservoir, the calibration liquid introduction flow path, and the calibration liquid discharge flow path are each hydrophilically treated.

10. The blood analyzer according to any one of claims 1 to 9, wherein the calibration liquid discharge channel is a capillary valve.

11. The blood analyzer according to any one of claims 1 to 10, wherein the sensor is an electrochemical sensor.

12. The blood analyzer according to any one of claims 1 to 11, wherein a blood collection mechanism that stores collected blood can be attached to the blood introduction port.

1 3. Blood analysis method consisting of the following steps:

(1) A blood cell / plasma separation unit disposed on the lower end side of the substrate, and a blood cell fraction storage unit that deposits and stores a blood cell fraction when a centrifugal force is applied, and a blood cell storage unit A plasma fraction storage unit that is located above the unit and stores plasma. Hey blood cells. A plasma separation unit; a sensor unit having a sensor groove that is disposed on the upper end side of the substrate and that analyzes a test component; a blood cell, a plasma guide channel that communicates the plasma separation unit and the sensor unit; A blood inlet for introducing a whole blood sample into the plasma separation unit; a calibration liquid reservoir for storing a calibration liquid for calibrating the sensor; a calibration liquid waste reservoir for storing a calibration liquid after sensor calibration; a calibration liquid reservoir; Providing a blood analyzer having a calibration liquid introduction flow path communicating with the sensor groove; and a calibration liquid discharge flow path communicating with the sensor groove and the calibration liquid waste reservoir;

(2) Centrifuge the blood analyzer around the first centrifuge central axis located above the blood cell fraction container and below the upper end of the plasma fraction container, and remove the calibration solution in the calibration reservoir. Introduced into the sensor groove;

(3) calibrate the sensor;

(4) Centrifuge the blood analyzer around the first centrifugal axis in the previous period, and discharge the calibration solution in the sensor groove to the calibration solution reservoir;

(6) centrifuge the blood analyzer around the first centrifugal axis to convey the plasma fractionated in the plasma fraction storage unit to the sensor groove;

(7) The sensor analyzes the liquid component in the plasma in the sensor groove.

14. The blood analysis method according to claim 13, wherein the gravitational acceleration applied to the sensor by centrifugation performed in steps (2), (4), and (6) is 500 G or less.

1 5. The gravity acceleration applied to the blood cell / plasma separator by centrifugation performed in step (5) is 1000 G or more, and the gravity acceleration applied to the sensor is 500 G or less. 1 3 blood analysis methods.

1 6. Blood analysis method comprising the following steps:

(1) A blood cell / plasma separation unit placed on the bottom side of the substrate, A blood cell fraction containing portion for precipitating and containing a blood cell fraction when acted thereon; a plasma fraction containing portion located above the blood cell fraction containing portion for containing plasma; and the plasma fraction containing portion A blood cell / plasma separation part having an air vent channel communicating with the sensor; a sensor part having a sensor groove disposed on the upper end side of the substrate and containing a sensor for analyzing a test component; and a blood cell / plasma separation part; A plasma guide channel communicating with the sensor unit; a blood introduction port for introducing a whole blood sample into the blood cell / plasma separation unit; a calibration liquid reservoir containing a calibration liquid for calibrating the sensor; a calibration liquid after sensor calibration A blood analyzer comprising: a calibration liquid waste reservoir that contains a calibration fluid; a calibration fluid introduction channel that communicates the calibration fluid reservoir and the sensor groove; and a calibration fluid discharge channel that communicates the sensor groove and the calibration fluid waste fluid reservoir Prepare;

(3) calibrate the sensor;

(6) The pressurized air is introduced from the air vent channel to convey the plasma fractionated in the plasma fraction storage unit to the sensor groove;

1 7. The blood analysis method according to claim 16, wherein the gravitational acceleration applied to the sensor by the centrifugation performed in steps (2) and (4) is 500 G or less.